Regimens for immunisation with meningococcal conjugates

ABSTRACT

Multivalent meningococcal conjugate vaccines are administered according to a schedule in which a first dose is administered to a patient aged between 0 and 12 months, and a second dose is administered to the patient aged between 12 and 24 months.

RELATED APPLICATIONS

All documents cited herein are incorporated by reference in theirentirety.

This application claims the benefit of U.S. Provisional Application No.60/785,234, filed Mar. 22, 2006, which is incorporated herein in itsentirety.

TECHNICAL FIELD

This invention is in the field of immunising patients with meningococcalconjugates.

BACKGROUND OF THE INVENTION

Conjugate vaccines for N. meningitidis serogroup C have been approvedfor human use, and include the products known as Menjugate™ [1],Meningitec™ and NeisVac-C™. Bivalent mixtures of conjugates fromserogroups A+C [2, 3] and C+Y [4] have also been reported. Mixtures ofconjugates from all four of serogroups A, C, W135 and Y are also known(e.g. see references 5-9), including the Menactra™ product that waslicensed in 2005.

In addition to the antigens included in a vaccine, an important aspectof effective immunisation is the dosing schedule. As noted in chapter 8of reference 10, “most vaccines require administration of multiple dosesin a primary series for development of immunity”. Moreover, “periodicrevaccination (‘booster doses’) with certain vaccines may be necessaryto maintain immunity”.

Known schedules for serogroup C meningococcal conjugate vaccinesinclude: a single dose at 12 months of age; two doses at 2 & 4 months;three doses at 2, 3 & 4 months of age; three doses at 2, 4 & 6 months ofage; three doses at 3, 5 & 12 months of age; three doses at 2, 4 & 12months. Alternative schedules, including the potential for a dose inlate infancy or the second year of life, have been suggested [11].

Multivalent meningococcal conjugate combinations have been administeredaccording to various dosing schedules. For example, known single-doseschedules for multivalent meningococcal conjugate vaccines include: at14 weeks of age [12]; at 6 months of age [13]; at 9 months [12]; between12-16 months [14]; between 2-3 years of age [5, 15]; between 2-10 years[16, 17, 18]; between 11-18 years [18]; 18-50 years [19]; 18-55 years[18]. The prescribing information for Menactra™ shows that it isadministered as a single dose in 11-18 or 18-55 year olds.

Known 2-dose schedules for multivalent meningococcal conjugate vaccinesinclude: 2 & 6 months of age [13]; first dose at 14 weeks of age, seconddose at 9 months of age [12]; first dose at 12-15 months, second dose 2months later [5]; first dose at 12-16 months, second dose 1 month later[14]; doses in 2 year olds at time zero and then 2 months later [18]; inadults at time zero and then 6 weeks later [2]; in adults at time zeroand then 2 months later [3]. A clinical study has also been reported inwhich patients received a first dose aged 11-18 years and a second dose3 years later.

Known 3-dose schedules for multivalent meningococcal conjugate vaccinesinclude: 6, 10 and 14 weeks of age [5, 12]; 2, 3 & 4 months [13]; 2, 4 &6 months of age [18]; 3, 4 & 5 months of age [20].

A 4-dose schedule at 6 weeks, 10 weeks, 14 weeks and 9 months isdisclosed in reference 12.

It is an object of the invention to provide further and improvedschedules for administering multivalent meningococcal conjugatevaccines, in particular to children.

SUMMARY OF THE INVENTION

The present invention relates to methods for immunizing a patient,comprising: (a) administering a multivalent meningococcal conjugatevaccine to the patient when they are aged between 0 and 12 months; and(b) administering a multivalent meningococcal conjugate vaccine to thepatient when they are aged between 12 and 24 months. The presentinvention also relates to methods for immunizing a patient whopreviously received a multivalent meningococcal conjugate vaccine to thepatient when they were aged between 0 and 12 months, comprising:administering a multivalent meningococcal conjugate vaccine to thepatient when they are aged between 12 and 24 months.

The invention further relates to kits comprising: (a) a multivalentmeningococcal conjugate vaccine; and (b) instructions for administeringthe vaccine according to a schedule that includes: (a) firstadministering the vaccine to a patient when they are aged between 0 and12 months; and (b) then administering the vaccine to a patient when theyare aged between 12 and 24 months.

The invention also relates to uses of a plurality of meningococcalconjugates in the manufacture of a medicament for administering to apatient in an immunisation schedule comprising: (a) administering themedicament to the patient when they are aged between 0 and 12 months;and (b) administering the medicament to the patient when they are agedbetween 12 and 24 months. The invention further relates to uses of aplurality of meningococcal conjugates in the manufacture of a medicamentfor administering to a patient who is aged between 12 and 24 months andwho previously received a multivalent meningococcal conjugate vaccinewhen they were aged between 0 and 12 months.

In one embodiment, the multivalent meningococcal conjugate vaccineincludes capsular saccharides from at least two of meningococcalserogroups A, C, W135 and Y. In a second embodiment, the multivalentmeningococcal conjugate vaccine includes capsular saccharides from allfour of meningococcal serogroups A, C, W135 and Y.

In another embodiment, a dose in the 0-12 month period and a dose in the12-24 month period are administered ≧6 months apart. In a furtherembodiment, a dose in the 0-12 month period is administered at 2 months,3 months, 4 months, 5 months or 6 months of age. In another embodiment,a dose in the 12-24 month period is administered at between 12-15 monthsof age or at between 15-18 months of age.

In a further embodiment, a dose in the 0-12 month period is administeredat substantially the same time as a hepatitis B virus vaccine, adiphtheria vaccine, a tetanus vaccine, a pertussis vaccine, aHaemophilus influenzae type b vaccine, a Streptococcus pneumoniaevaccine, and/or a polio vaccine. In another embodiment, a dose in the12-24 month period is administered at substantially the same time as ahepatitis B virus vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a Haemophilus influenzae type b vaccine, aStreptococcus pneumoniae vaccine, a polio vaccine, an influenza vaccine,a chickenpox vaccine, a measles vaccine, a mumps vaccine, and/or arubella vaccine.

In a particular embodiment, the multivalent meningococcal conjugatevaccine has a carrier protein selected from the group consisting ofdiphtheria toxoid, tetanus toxoid and CRM197. In another embodiment, themultivalent meningococcal conjugate vaccine is unadjuvanted.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, multivalent meningococcal conjugate vaccinesare administered according to a schedule in which a first dose isadministered to a patient aged between 0 and 12 months, and a seconddose is administered to a patient aged between 12 and 24 months. Thisschedule offers early protection than the existing licensed schedule,reduces the cost of immunisation by avoiding the need for a thirdimmunisation, and the second dose can act as a booster dose forproviding long-lasting protection.

Thus the invention provides a method for immunising a patient,comprising: (a) administering a multivalent meningococcal conjugatevaccine to the patient when they are aged between 0 and 12 months; and(b) administering a multivalent meningococcal conjugate vaccine to thepatient when they are aged between 12 and 24 months.

The invention also provides a method for immunising a patient whopreviously received a multivalent meningococcal conjugate vaccine to thepatient when they were aged between 0 and 12 months, comprising:administering a multivalent meningococcal conjugate vaccine to thepatient when they are aged between 12 and 24 months.

The invention also provides the use of a plurality of meningococcalconjugates in the manufacture of a medicament for administering to apatient in an immunisation schedule comprising: (a) administering themedicament to the patient when they are aged between 0 and 12 months;and (b) administering the medicament to the patient when they are agedbetween 12 and 24 months.

The invention also provides the use of a plurality of meningococcalconjugates in the manufacture of a medicament for administering to apatient who is aged between 12 and 24 months and who previously receiveda multivalent meningococcal conjugate vaccine when they were agedbetween 0 and 12 months.

The invention also provides a kit comprising: (a) a multivalentmeningococcal conjugate vaccine; and (b) instructions for administeringthe vaccine according to a schedule that includes: (a) firstadministering the vaccine to a patient when they are aged between 0 and12 months; and (b) then administering the vaccine to a patient when theyare aged between 12 and 24 months.

The Schedule

The schedule of the invention involves a first dose in the first year oflife and a second dose in the second year of life. The first dose isgiven to a patient aged between 0 and 12 months, up to but not includingtheir first birthday. The second dose is given to a patient aged 12 and24 months, starting on the day of their first birthday, up to andincluding their second birthday.

Within this overall schedule, the two doses can be administered at anytime. In general, however, the two doses will be administered at least 4weeks apart e.g. ≧8 weeks apart, ≧2 months apart, ≧3 months apart, ≧6months apart, etc.

Within the 0-12 month period, the first dose is preferably notadministered before about 6 weeks of age. after 5 weeks. Typical timesfor receiving the first dose are at 2 months, 3 months, 4 months, 5months or 6 months of age.

Within the 12-24 month period, the second dose is preferablyadministered in the first half i.e. between 12 and 18 months e.g.between 12 and 15 months of age, or between 15 and 18 months.

The patient will not have received a meningococcal conjugate vaccinebefore the first dose in the schedule. In preferred embodiments, thepatient does not receive a meningococcal conjugate vaccine between thefirst dose and the second dose, but sometimes an intermediate dose maybe administered. For example, the patient may receive 2 or 3 doses inthe 0-12 month period e.g. at 2, 3 & 4 months of age, at 3, 4 & 5 monthsof age, at 2, 4 & 6 months, at 3, 5 & 9 months etc.

In some embodiments, the patient does not receive a further dose, but inother embodiments they can do so. Such a further dose is preferably notadministered until after the patient's second birthday e.g. until aftertheir fifth birthday, after their tenth birthday, after their fifteenthbirthday, after their seventeenth birthday, after their twenty-firstbirthday, etc. The further dose may be administered when circulatingantibody levels have declined to undetectable levels [21].

Conveniently, the first dose can be administered at substantially thesame time as (e.g. during the same medical consultation or visit to ahealthcare professional) another vaccine e.g. at substantially the sametime as a hepatitis B virus vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine (either cellular or, preferably,acellular), a Haemophilus influenzae type b vaccine, a Streptococcuspneumoniae vaccine, and/or a polio vaccine (preferably in inactivatedpoliovirus vaccine). Each of these optionally co-administered vaccinesmay be a monovalent vaccine or may be part of a combination vaccine(e.g. as part of a D-T-P vaccine).

Conveniently, the second dose can be administered at substantially thesame time as (e.g. during the same medical consultation or visit to ahealthcare professional) another vaccine e.g. at substantially the sametime as a hepatitis B virus vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine (either cellular or acellular), aHaemophilus influenzae type b vaccine, a Streptococcus pneumoniaevaccine, a polio vaccine (preferably in inactivated poliovirus vaccine),an influenza vaccine, a chickenpox vaccine, a measles vaccine, a mumpsvaccine, and/or a rubella vaccine. Each of these optionallyco-administered vaccines may be a monovalent vaccine or may be part of acombination vaccine (e.g. as part of a M-M-R vaccine).

The Vaccine

The invention involves the administration of multivalent meningococcalconjugate vaccines i.e. vaccines that, when administered, simultaneouslyprovide immunity against 2, 3, 4 or more different serotypes of N.meningitidis. Multivalent vaccines against 2, 3, or 4 of serogroups A,C, W135 and Y are preferred e.g. A+C, C+Y, W135+Y, A+W135+Y, A+C+W135+Y,etc. Vaccines including at least serogroup C are preferred (e.g. A+C),and vaccines including saccharides from all four of serogroups A, C,W135 and Y are most preferred.

The vaccines include a meningococcal capsular saccharide conjugated to acarrier protein.

The capsular saccharide of serogroup A meningococcus is a homopolymer of(α1→6)-linked N-acetyl-D-mannosamine-1-phosphate, with partialO-acetylation in the C3 and C4 positions. The acetyl groups can bereplaced with blocking groups to prevent hydrolysis [22], and suchmodified saccharides are still serogroup A saccharides within themeaning of the present invention. The serogroup C capsular saccharide isa homopolymer of (α2→9)-linked sialic acid (N-acetyl neuraminic acid, or‘NeuNAc’). Most serogroup C strains have O-acetyl groups at C-7 and/orC-8 of the sialic acid residues, but about 15% of clinical isolates lackthese O-acetyl groups [23, 24]. The saccharide structure is written as→9)-Neu p NAc 7/8 OAc-(α2→. The serogroup W135 saccharide is a polymerof sialic acid-galactose disaccharide units. Like the serogroup Csaccharide, it has variable O-acetylation, but at sialic acid 7 and 9positions [25]. The structure is written as:→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α-(1→. The serogroup Y saccharide issimilar to the serogroup W135 saccharide, except that the disacchariderepeating unit includes glucose instead of galactose. Like serogroupW135, it has variable O-acetylation at sialic acid 7 and 9 positions[25]. The serogroup Y structure is written as:→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Glc-α-(1→.

The saccharides used according to the invention may be O-acetylated asdescribed above (e.g. with the same O-acetylation pattern as seen innative capsular saccharides), or they may be partially or totallyde-O-acetylated at one or more positions of the saccharide rings, orthey may be hyper-O-acetylated relative to the native capsularsaccharides. Serogroup C saccharides used with the invention may beprepared from either OAc+ or OAc− strains. Preferred strains forproduction of serogroup C conjugates are OAc+ strains, preferably ofserotype 16, preferably of serosubtype P1.7a,1. Thus C: 16:P1.7a,1 OAc+strains are preferred. Preferably at least 50% (e.g. at least 60%, 70%,80%, 90%, 95% or more) of the mannosamine residues in a serogroup Asaccharides are O-acetylated at the C-3 position.

The saccharide moieties in conjugates may comprise full-lengthsaccharides as prepared from meningococci, and/or it may comprisefragments of full-length saccharides. The saccharides used according tothe invention are preferably shorter than the native capsularsaccharides seen in bacteria. Thus the saccharides are preferablydepolymerised, with depolymerisation occurring during or aftersaccharide purification but before conjugation. Depolymerisation reducesthe chain length of the saccharides. One depolymerisation methodinvolves the use of hydrogen peroxide [5]. Hydrogen peroxide is added toa saccharide (e.g. to give a final H₂O₂ concentration of 1%), and themixture is then incubated (e.g. at about 55° C.) until a desired chainlength reduction has been achieved. Another depolymerisation methodinvolves acid hydrolysis [5]. Other depolymerization methods are knownin the art. The saccharides used to prepare conjugates for use accordingto the invention may be obtainable by any of these depolymerisationmethods. Depolymerisation can be used in order to provide an optimumchain length for immunogenicity and/or to reduce chain length forphysical manageability of the saccharides. Preferred saccharides havethe following range of average degrees of polymerisation (Dp): A=10-20;C=12-22; W135=15-25; Y=15-25. In terms of molecular weight, rather thanDp, preferred ranges are, for all serogroups: <100 kDa; 5 kDa-75 kDa; 7kDa-50 kDa; 8 kDa-35 kDa; 12 kDa-25 kDa; 15 kDa-22 kDa.

Typical carrier proteins for use in conjugates are bacterial toxins,such as diphtheria toxin [e.g. see chapter 13 of ref. 10; refs. 26-29](or its CRM197 mutant [30-33]) and tetanus toxin, usually in toxoid form(e.g. obtained by treatment with an inactivating chemical, such asformalin or formaldehyde). Other suitable carrier proteins include, butare not limited to, N. meningitidis outer membrane protein [34],synthetic peptides [35, 36], heat shock proteins [37, 38], pertussisproteins [39, 40], cytokines [41], lymphokines [41], hormones [41],growth factors [41], artificial proteins comprising multiple human CD4⁺T cell epitopes from various pathogen-derived antigens [42] such as N19[43], protein D from H. influenzae [44-46], pneumolysin [47],pneumococcal surface protein PspA [48], iron-uptake proteins [49], toxinA or B from C. difficile [50], etc.

Four particularly preferred carrier proteins are diphtheria toxoid (Dt),tetanus toxoid (Tt), CRM197 and protein D from H. influenzae. Theseproteins are preferred because they are the main carriers currently inuse in pediatric vaccines e.g. the Hib conjugates from GSK use Tt as thecarrier, the HibTITER™ product uses CRM197, the pneumococcal conjugatesin Prevenar™ use CRM197, the Menjugate™ and Meningitec™ products useCRM197, and NeisVac-C™ uses Tt.

Conjugates are preferably mixed at substantially equal masses (measuredas mass of saccharide) e.g. the mass of each serogroup's saccharide iswithin ±10% of each other. A typical quantity of meningococcal antigenper serogroup in a composition is between 1 μg and 20 μg e.g. between 2and 10 μg per serogroup, or about 4 μg. As an alternative to an equalratio, a double serogroup A dose may be used.

Conjugates with a saccharide:protein ratio (w/w) of between 1:15 (i.e.excess protein) and 15:1 (i.e. excess saccharide), preferably between1:5 and 5:1, are preferred. Excess carrier protein is preferred.Conjugates with saccharide:protein ratio of about 1:12 or about 1:3 arepreferred, particularly where the carrier is Dt.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalised prior toconjugation. Activation may involve, for example, cyanylating reagents[51, 52, etc.]). Other suitable techniques use active esters,carbodiimides, hydrazides, norborane, p-nitrobenzoic acid,N-hydroxysuccinimide, S—NHS, EDC, TSTU; see also the introduction toreference 53).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 54 and 55. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group[56, 57, 58]. Other linkers include B-propionamido [59],nitrophenyl-ethylamine [60], haloacyl halides [61], glycosidic linkages[62], 6-aminocaproic acid [63], ADH [64], C₄ to C₁₂ moieties [65] etc.As an alternative to using a linker, direct linkage can be used. Directlinkages to the protein may comprise oxidation of the polysaccharidefollowed by reductive amination with the protein, as described in, forexample, references 66 and 67.

A preferred conjugation process involves: introduction of amino groupsinto the saccharide (e.g. by replacing terminal ═O groups with —NH₂)followed by derivatisation with an adipic diester (e.g. adipic acidN-hydroxysuccinimide diester) and reaction with carrier protein (e.g.CRM197). Further details of this conjugation method can be found inreference 6. Conjugates obtainable by this method are preferredconjugates for use according to the invention.

In another preferred conjugation process, a saccharide is reacted withadipic acid dihydrazide. For serogroup A, carbodiimide (EDAC) may alsobe added at this stage. After a reaction period, sodium cyanoborohydrideis added. Derivatised saccharide can then be prepared e.g. byultrafiltration. The derivatized saccharide is then mixed with carrierprotein (e.g. with a diphtheria toxoid), and carbodiimide is added.After a reaction period, the conjugate can be recovered. Further detailsof this conjugation method can be found in reference 6. Conjugatesobtainable by this method are preferred conjugates for use according tothe invention e.g. conjugates comprising a diphtheria toxoid carrier andan adipic acid linker.

In another preferred conjugation process, a saccharide is derivatisedwith a cyanylating reagent [52], followed by coupling to a protein(direct, or after introduction of a thiol or hydrazide nucleophile groupinto the carrier), without the need to use a linker. Suitablecyanylating reagents include 1-cyano-4-(dimethylamino)-pyridiniumtetrafluoroborate (‘CDAP’), p-nitrophenylcyanate andN-cyanotriethylammonium tetrafluoroborate (‘CTEA’). CDAP is preferred,particularly where H. influenzae protein D is the common carrier. Directcoupling is preferred.

Administration of a conjugate preferably results in an increase in serumbactericidal assay (SBA) titre for the relevant serogroup of at least4-fold, and preferably at least 8-fold, measured with human complement[68]. If rabbit complement is used to measure SBA titres then the titreincrease is preferably at least 128-fold.

Conjugates are preferably prepared separately and then mixed. Thus it ispreferred not to use a single protein carrying multiple serogroups (cf.references 69 & 70). After mixing, the concentration of the mixedconjugates can be adjusted e.g. with sterile pyrogen-free,phosphate-buffered saline.

In compositions of the invention, the amount of carrier (conjugated andunconjugated) from each conjugate is preferably no more than 100 μg/mle.g. <30 μg/ml of carrier protein from each conjugate. Preferredcompositions include a total concentration of carrier (either solely forthe combined meningococcal conjugates, or preferably for the compositionas a whole) of less than 500 μg/ml e.g. <400 μg/ml, <300 μg/ml, <200μg/ml, <100 μg/ml, <50 μg/ml, etc.

Vaccines of the invention may include no antigens other than themeningococcal conjugates. In some embodiments, however, vaccines mayinclude further antigens. Thus they may include further antigens fromother pathogens, particularly from bacteria and/or viruses. They mayinclude other conjugated saccharides from non-meningococcal organismsand/or they may include non-saccharide antigens. For example, they mayinclude one or more of the following:

-   -   a diphtheria toxoid (‘D’).    -   a tetanus toxoid (‘T’).    -   a pertussis antigen (‘P’), which is typically acellular (‘aP’).    -   a hepatitis B virus (HBV) surface antigen (‘HBsAg’).    -   a hepatitis A virus (HAV) antigen.    -   a conjugated Haemophilus influenzae type b capsular saccharide        (‘Hib’).    -   a protein from serogroup B of N. meningitidis.    -   an vesicle preparation from serogroup B of N. meningitidis.    -   inactivated poliovirus vaccine (IPV).

The schedule of the invention may use different vaccines for the firstand second doses e.g. the first vaccine may include non-meningococcalantigens whereas the second vaccine does not, or the first vaccine mayinclude a first set of non-meningococcal antigens (e.g. DTP) whereas thesecond vaccine includes a second (different) set of non-meningococcalantigens (e.g. MMR).

In addition to the antigenic components described above, compositions ofthe invention will generally include a non-antigenic component. Thenon-antigenic component can include carriers, adjuvants, excipients,buffers, etc., as described in more detail below. These non-antigeniccomponents may have various sources. For example, they may be present inone of the antigen or adjuvant materials that is used during manufactureor may be added separately from those components. Preferred compositionsof the invention include one or more pharmaceutical carrier(s) and/orexcipient(s). A thorough discussion of pharmaceutically acceptablecarriers and excipients is available in reference 71.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [72], but keeping osmolality in this range is neverthelesspreferred.

Compositions of the invention may include one or more buffers. Typicalbuffers include: a phosphate buffer; a Tris buffer; a borate buffer; asuccinate buffer; a histidine buffer; or a citrate buffer. Buffers willtypically be included in the 5-20 mM range.

The pH of a composition of the invention will generally be between 5.0and 7.5, and more typically between 5.0 and 6.0 for optimum stability,or between 6.0 and 7.0.

Compositions of the invention are preferably sterile.

Compositions of the invention are preferably non-pyrogenic e.g.containing <1 EU (endotoxin unit, a standard measure) per dose, andpreferably <0.1 EU per dose.

Compositions of the invention are preferably gluten free.

Where antigens are adsorbed, a composition may be a suspension with acloudy appearance. This appearance means that microbial contamination isnot readily visible, and so the vaccine preferably contains apreservative. This is particularly important when the vaccine ispackaged in multidose containers. Preferred preservatives for inclusionare 2-phenoxyethanol and thimerosal. It is recommended, however, not touse mercurial preservatives (e.g. thimerosal) where possible. It ispreferred that compositions of the invention contain less than about 25ng/ml mercury.

The concentration of any aluminium salts in a composition of theinvention, expressed in terms of Al³⁺, is preferably less than 5 mg/mle.g. ≦4 mg/ml, ≦3 mg/ml, ≦2 mg/ml, ≦1 mg/ml, etc.

Compositions of the invention are preferably administered to patients in0.5 ml doses. References to 0.5 ml doses will be understood to includenormal variance e.g. 0.5 ml±0.05 ml.

Residual material from individual antigenic components may also bepresent in trace amounts in the final vaccine produced by the process ofthe invention. For example, if formaldehyde is used to prepare thetoxoids of diphtheria, tetanus and pertussis then the final vaccineproduct may retain trace amounts of formaldehyde (e.g. less than 10μg/ml, preferably <5 μg/ml). Media or stabilizers may have been usedduring poliovirus preparation (e.g. Medium 199), and these may carrythrough to the final vaccine. Similarly, free amino acids (e.g. alanine,arginine, aspartate, cysteine and/or cystine, glutamate, glutamine,glycine, histidine, proline and/or hydroxyproline, isoleucine, leucine,lysine, methionine, phenylalanine, serine, threonine, tryptophan,tyrosine and/or valine), vitamins (e.g. choline, ascorbate, etc.),disodium phosphate, monopotassium phosphate, calcium, glucose, adeninesulfate, phenol red, sodium acetate, potassium chloride, etc. may beretained in the final vaccine at ≦100 μg/ml, preferably <10 μg/ml, each.Other components from antigen preparations, such as neomycin (e.g.neomycin sulfate, particularly from an IPV component), polymyxin B (e.g.polymyxin B sulfate, particularly from an IPV component), etc. may alsobe present e.g. at sub-nanogram amounts per dose.

A further possible component of the final vaccine which originates inthe antigen preparations arises from less-than-total purification ofantigens. Small amounts of B. pertussis, C. diphtheriae, C. tetaniand/or S. cerevisiae proteins and/or genomic DNA may therefore bepresent.

Meningococcal conjugates may be lyophilised prior to use according tothe invention. If lyophilised, the composition may include a stabilisersuch as mannitol. It may also include sodium chloride.

The Patient

The age of patients receiving vaccines of the invention is dictated bythe schedule.

Although the patient will not have received a meningococcal conjugatevaccine before the first dose in the schedule, they may have receivedother non-meningococcal conjugates and/or they may have received thecarrier protein that is used in the meningococcal conjugate. Priorexposure to the carrier may have been as carrier in non-meningococcalconjugate (e.g. in a Hib conjugate) and/or as antigen itself (e.g.tetanus toxoid is commonly used as carrier for Hib conjugates, but isalso used as an antigen for protecting against C. tetani).

After receiving the first dose in the schedule, and before the seconddose, a patient is distinguishable from a person in the generalpopulation, as they will have mounted an immune response against thefirst dose. Thus patients waiting to receive the schedule's second doseare a specific and identifiable subset of the population.

Compositions of the invention can be administered by intramuscularinjection e.g. into the arm, leg or buttock. Where another vaccine isco-administered then it is typical to inject compositions into oppositelimbs e.g. to inject one into the left arm and one into the right arm.

Where compositions of the invention include an aluminium-based adjuvant,settling of components may occur during storage. The composition shouldtherefore be shaken prior to administration to a patient. The shakencomposition will generally be a turbid white suspension.

The patient is a human.

Packaging

Vaccines for use with the invention can be placed into containers foruse. Suitable containers include vials and disposable syringes(preferably sterile ones).

Where a composition of the invention is packaged into vials, these arepreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Whenusing a multidose vial, each dose should be withdrawn with a sterileneedle and syringe under strict aseptic conditions, taking care to avoidcontaminating the vial contents. Preferred vials are made of colorlessglass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilised materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed.

Where the composition is packaged into a syringe, the syringe will notnormally have a needle attached to it, although a separate needle may besupplied with the syringe for assembly and use. Safety needles arepreferred. 1-inch 23-gauge, 1-inch 25-gauge and ⅝-inch 25-gauge needlesare typical. Syringes may be provided with peel-off labels on which thelot number and expiration date of the contents may be printed, tofacilitate record keeping. The plunger in the syringe preferably has astopper to prevent the plunger from being accidentally removed duringaspiration. The syringes may have a latex rubber cap and/or plunger.Disposable syringes contain a single dose of vaccine. The syringe willgenerally have a tip cap to seal the tip prior to attachment of aneedle, and the tip cap is preferably made of butyl rubber. If thesyringe and needle are packaged separately then the needle is preferablyfitted with a butyl rubber shield. Grey butyl rubber is preferred.Preferred syringes are those marketed under the trade name “Tip-Lok”™.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

If a vaccine is in lyophilised form then it will usually be resuspendedinto an aqueous form prior to administration.

In addition to containing vaccines for administration, kits of theinvention can include instructions for administering the vaccine. Theinstructions will refer to an immunisation schedule that includes: (a)first administering the vaccine to a patient when they are aged between0 and 12 months; and (b) then administering the vaccine to a patientwhen they are aged between 12 and 24 months.

Adjuvants

Vaccines of the invention may include an adjuvant. Where a vaccineincludes only meningococcal conjugates, however, use of an adjuvant isnot preferred. Where an adjuvant is used, it may comprise one or morealuminium salts, and particularly an aluminium phosphate adjuvant and/oran aluminium hydroxide adjuvant.

Aluminium adjuvants currently in use are typically referred to either as“aluminium hydroxide” or as “aluminium phosphate” adjuvants. These arenames of convenience, however, as neither is a precise description ofthe actual chemical compound which is present (e.g. see chapter 9 ofreference 73). The invention can use any of the “hydroxide” or“phosphate” salts that are in general use as adjuvants.

The adjuvants known as “aluminium hydroxide” are typically aluminiumoxyhydroxide salts, which are usually at least partially crystalline.Aluminium oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminium compounds, such as aluminiumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ (chapter 9 of ref. 73).

The adjuvants known as “aluminium phosphate” are typically aluminiumhydroxyphosphates, often also containing a small amount of sulfate. Theymay be obtained by precipitation, and the reaction conditions andconcentrations during precipitation can influence the degree ofsubstitution of phosphate for hydroxyl in the salt. Hydroxyphosphatesgenerally have a PO₄/Al molar ratio between 0.3 and 0.99.Hydroxyphosphates can be distinguished from strict AlPO₄ by the presenceof hydroxyl groups. For example, an IR spectrum band at 3164 cm⁻¹ (e.g.when heated to 200° C.) indicates the presence of structural hydroxyls(chapter 9 of ref. 73).

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate. Typical diameters of the particles are in therange 0.5-20 μm (e.g. about 5-10 μm) after any antigen adsorption.

The PZC of aluminium phosphate is inversely related to the degree ofsubstitution of phosphate for hydroxyl, and this degree of substitutioncan vary depending on reaction conditions and concentration of reactantsused for preparing the salt by precipitation. PZC is also altered bychanging the concentration of free phosphate ions in solution (morephosphate=more acidic PZC) or by adding a buffer such as a histidinebuffer (makes PZC more basic). Aluminium phosphates used according tothe invention will generally have a PZC of between 4.0 and 7.0, morepreferably between 5.0 and 6.5 e.g. about 5.7.

An aluminium phosphate solution used to prepare a composition of theinvention may contain a buffer (e.g. a phosphate or a histidine or aTris buffer), but this is not always necessary. The aluminium phosphatesolution is preferably sterile and pyrogen-free. The aluminium phosphatesolution may include free aqueous phosphate ions e.g. present at aconcentration between 1.0 and 20 mM, preferably between 5 and 15 mM, andmore preferably about 10 mM. The aluminium phosphate solution may alsocomprise sodium chloride. The concentration of sodium chloride ispreferably in the range of 0.1 to 100 mg/ml (e.g. 0.5-50 mg/ml, 1-20mg/ml, 2-10 mg/ml) and is more preferably about 3±1 mg/ml. The presenceof NaCl facilitates the correct measurement of pH prior to adsorption ofantigens.

A mixture of both an aluminium hydroxide adjuvant and an aluminiumphosphate adjuvant can be used. If so, there may be more aluminiumphosphate than hydroxide e.g. a weight ratio of at least 2:1 e.g. ≧5:1,≧6:1, ≧7:1, ≧8:1, ≧9:1, etc.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y. The word “substantially” does notexclude “completely” e.g. a composition which is “substantially free”from Y may be completely free from Y. Where necessary, the word“substantially” may be omitted from the definition of the invention.

The term “about” in relation to a numerical value x means, for example,x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where an antigen is described as being “adsorbed” to an adjuvant, it ispreferred that at least 50% (by weight) of that antigen is adsorbed e.g.50%, 60%, 70%, 80%, 90%, 95%, 98% or more. It is preferred thatdiphtheria toxoid and tetanus toxoid are both totally adsorbed i.e. noneis detectable in supernatant. Total adsorption of HBsAg is alsopreferred.

Amounts of conjugates are generally given in terms of mass of saccharide(i.e. the dose of the conjugate (carrier+saccharide) as a whole ishigher than the stated dose) in order to avoid variation due to choiceof carrier.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE).

EXAMPLE

The immunogenicity, safety, tolerability and the ability to prime formemory of a meningococcal conjugate vaccine are investigated in amulti-centre, open-label, controlled, randomized study. Infants aresplit into three groups to receive an unadjuvanted 4-valent conjugatedA-C-W135-Y vaccine as follows, with the group 1 schedule being anembodiment of the invention:

-   -   1: first doses at about 6 months, then a second dose at about 12        months (on or after birthday)    -   2: single dose at about 12 months (on or after birthday)    -   3: dose of monovalent MenC at 12 months, then 4-valent at 18        months.

Meningococcal conjugates are administered at the same time as otherroutine pediatric vaccines, and blood samples for serological analysisare taken both at the time of vaccination and 1 month later: Visit 1Visit 2 Visit 3 Visit 4 Group 1 6 months 7 months 12 months 13 months B,M4, PC7, 5 B B, M4, PC7 B, 4V Group 2 6 months 7 months 12 months 73months B, PC7, 5 B B, M4, PC7 B, 4V Group 3 12 months 13 months 18months 19 months B, M1, PC7 B, 4V B, M4, 5 BKey: B = blood taken for serology; 5 = D-T-Pa-Hib-IPV; PC7 = 7-valentpneumococcal conjugate; 4V = MMR + V; M4 = 4-valent Men-A-C-W135-Yconjugates; M1 = Men-C conjugate.

Immunogenicity is assessed by evaluating serum antibody responses bymeasuring bactericidal antibody titers.

For blood samples taken at the first 2 visits, the bactericidal antibodytiter at visit 2, expressed as a ratio relative to visit 1, was asfollows for each group: A C W135 Y Group 1 1.5 11 2.8 1.8 Group 2 1.0 11.0 1.0 Group 3 1.0 20 1.0 1.0

It will be understood that the invention has been described by way ofexample only, and that modifications may be made whilst remaining withinthe scope and spirit of the invention.

REFERENCES The Contents of which are Hereby Incorporated by Reference

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1. A method for immunizing a patient, comprising: (a) administering amultivalent meningococcal conjugate vaccine to the patient when they areaged between 0 and 12 months; and (b) administering a multivalentmeningococcal conjugate vaccine to the patient when they are agedbetween 12 and 24 months.
 2. The method of claim 1, where themultivalent meningococcal conjugate vaccine includes capsularsaccharides from at least two of meningococcal serogroups A, C, W135 andY.
 3. The method of claim 2, where the multivalent meningococcalconjugate vaccine includes capsular saccharides from all four ofmeningococcal serogroups A, C, W135 and Y.
 4. The method of claim 1,wherein a dose in the 0-12 month period and a dose in the 12-24 monthperiod are administered ≧6 months apart.
 5. The method of claim 1,wherein a dose in the 0-12 month period is administered at 2 months, 3months, 4 months, 5 months or 6 months of age.
 6. The method of claim 1,wherein a dose in the 12-24 month period is administered at between12-15 months of age or at between 15-18 months of age.
 7. The method ofclaim 1, wherein a dose in the 0-12 month period is administered atsubstantially the same time as a hepatitis B virus vaccine, a diphtheriavaccine, a tetanus vaccine, a pertussis vaccine, a Haemophilusinfluenzae type b vaccine, a Streptococcus pneumoniae vaccine, and/or apolio vaccine.
 8. The method of claim 1, wherein a dose in the 12-24month period is administered at substantially the same time as ahepatitis B virus vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a Haemophilus influenzae type b vaccine, aStreptococcus pneumoniae vaccine, a polio vaccine, an influenza vaccine,a chickenpox vaccine, a measles vaccine, a mumps vaccine, and/or arubella vaccine.
 9. The method of claim 1, wherein the multivalentmeningococcal conjugate vaccine has a carrier protein selected from thegroup consisting of diphtheria toxoid, tetanus toxoid and CRM197. 10.The method of claim 1, wherein the multivalent meningococcal conjugatevaccine is unadjuvanted.
 11. A method for immunizing a patient whopreviously received a multivalent meningococcal conjugate vaccine to thepatient when they were aged between 0 and 12 months, comprising:administering a multivalent meningococcal conjugate vaccine to thepatient when they are aged between 12 and 24 months.
 12. The method ofclaim 11, where the multivalent meningococcal conjugate vaccine includescapsular saccharides from at least two of meningococcal serogroups A, C,W135 and Y.
 13. The method of claim 12, where the multivalentmeningococcal conjugate vaccine includes capsular saccharides from allfour of meningococcal serogroups A, C, W135 and Y.
 14. The method ofclaim 11, wherein a dose in the 0-12 month period and a dose in the12-24 month period are administered ≧6 months apart.
 15. The method ofclaim 11, wherein a dose in the 0-12 month period is administered at 2months, 3 months, 4 months, 5 months or 6 months of age.
 16. The methodof claim 11, wherein a dose in the 12-24 month period is administered atbetween 12-15 months of age or at between 15-18 months of age.
 17. Themethod of claim 11, wherein a dose in the 0-12 month period isadministered at substantially the same time as a hepatitis B virusvaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, aHaemophilus influenzae type b vaccine, a Streptococcus pneumoniaevaccine, and/or a polio vaccine.
 18. The method of claim 11, wherein adose in the 12-24 month period is administered at substantially the sametime as a hepatitis B virus vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a Haemophilus influenzae type b vaccine, aStreptococcus pneumoniae vaccine, a polio vaccine, an influenza vaccine,a chickenpox vaccine, a measles vaccine, a mumps vaccine, and/or arubella vaccine.
 19. The method of claim 11, wherein the multivalentmeningococcal conjugate vaccine has a carrier protein selected from thegroup consisting of diphtheria toxoid, tetanus toxoid and CRM197. 20.The method of claim 11, wherein the multivalent meningococcal conjugatevaccine is unadjuvanted.
 21. A kit comprising: (a) a multivalentmeningococcal conjugate vaccine; and (b) instructions for administeringthe vaccine according to a schedule that includes: (a) firstadministering the vaccine to a patient when they are aged between 0 and12 months; and (b) then administering the vaccine to a patient when theyare aged between 12 and 24 months.
 22. The kit of claim 21, where themultivalent meningococcal conjugate vaccine includes capsularsaccharides from at least two of meningococcal serogroups A, C, W135 andY.
 23. The kit of claim 22, where the multivalent meningococcalconjugate vaccine includes capsular saccharides from all four ofmeningococcal serogroups A, C, W135 and Y.
 24. The kit of claim 21,wherein a dose in the 0-12 month period and a dose in the 12-24 monthperiod are administered ≧6 months apart.
 25. The kit of claim 21,wherein a dose in the 0-12 month period is administered at 2 months, 3months, 4 months, 5 months or 6 months of age.
 26. The kit of claim 21,wherein a dose in the 12-24 month period is administered at between12-15 months of age or at between 15-18 months of age.
 27. The kit ofclaim 21, wherein a dose in the 0-12 month period is administered atsubstantially the same time as a hepatitis B virus vaccine, a diphtheriavaccine, a tetanus vaccine, a pertussis vaccine, a Haemophilusinfluenzae type b vaccine, a Streptococcus pneumoniae vaccine, and/or apolio vaccine.
 28. The kit of claim 21, wherein a dose in the 12-24month period is administered at substantially the same time as ahepatitis B virus vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a Haemophilus influenzae type b vaccine, aStreptococcus pneumoniae vaccine, a polio vaccine, an influenza vaccine,a chickenpox vaccine, a measles vaccine, a mumps vaccine, and/or arubella vaccine.
 29. The kit of claim 21, wherein the multivalentmeningococcal conjugate vaccine has a carrier protein selected from thegroup consisting of diphtheria toxoid, tetanus toxoid and CRM197. 30.The kit of claim 21, wherein the multivalent meningococcal conjugatevaccine is unadjuvanted.